Metabolic controlled fermentation procedure for the manufacture of lovastatin hydroxy acid

ABSTRACT

A method for producing mevinolin by a microorganism in a fermentation process having a seed culture stage and a main fermentation stage, including 
     a) cultivating a microorganism biomass in the seed culture stage to produce an inoculum; 
     b) transferring the inoculum into a fermentation medium in the main fermentation stage; and, 
     c) maintaining steady stage conditions in the main fermentation stage, thereby producing a fermentation broth containing mevinolin. Preferably, the steady state conditions are maintained in the main fermentation stage by one or more of feeding of organic carbon sources; controlling glucose and/or total reducing sugar content; feeding of organic nitrogen sources; controlling pH; controlling foam level; controlling the mass of the fermentation broth by withdrawals and feedings; and, controlling the dissolved oxygen level.

FIELD OF THE INVENTION

This invention relates generally to the biosynthesis of cholesterolreducing agents. More specifically, the invention relates to thebiosynthesis of the cholesterol lowering agent mevinolin by certainmicroorganisms.

BACKGROUND OF THE INVENTION

Mevinolin (lovastatin; monacolin K;β,δ-dihydroxy-7-[1,2,6,7,8,8a-hexahydro-2,6-dimethyl-8-(2-methyl-butyryloxy)-naphtalen-1-yl]-heptanoicacid δ-lactone) is one of the most important known cholesterol loweringagents. Mevinolin, as used herein, includes both the lactone and freehydroxy acid forms.

Its open hydroxy acid form is a potent inhibitor of the3-hydroxy-3-methyl-glutarylcoenzyme A reductase enzyme, which catalysesthe formation of mevalonic acid, an early intermediate of cholesterolbiosynthesis. Mevinolin is specifically advantageous because, as aresult of its application, biosynthetic intermediates with a toxicsteroid skeleton, formed at a later stage of biosynthesis fail toaccumulate. Mevinolin increases the number of LDL-receptors at thesurface of the cell membrane which remove the LDL cholesterolcirculating in the blood, thereby inducing the lowering of blood plasmacholesterol level.

Commonly, the active ingredient is produced via fermentation. GB 2046737discloses that the active ingredient can be produced by some strainsbelonging to the Monascus genus e.g. by M. ruber 1005 cultivated between7 and 40° C. As a culture medium the aqueous solution of glucose,peptone, corn steep liquor and ammonium chloride was used. Thefermentation was carried out for 10 days in aerobic conditions, and 87mg mevinolin was obtained from the filtrate of 5 liters broth.

U.S. Pat. No. 4,294,926 discloses the biosynthesis of the mevinolinpreferably by the application of microorganisms under the depositednumbers ATCC 20541 or 20542 belonging to the Aspergillus terreus genuson a culture medium containing carbohydrates, e.g., glucose, fructose,maltose, as carbon source, nitrogen sources, e.g., yeast, hydrolyzedyeast, hydrolyzed casein, corn steep liquor; and mineral salts, e.g.,calcium carbonate, magnesium sulphate, cobalt, ferro, manganic salts ata temperature of 20-37° C. Similar procedures are described in U.S. Pat.Nos. 4,420,491, 4,342,767, 4,319,039 and 4,294,846, where thefermentations are carried out for 3-5 days on media containing 1-6%carbohydrates and 0.2-6% nitrogen sources.

German Patent No. 4,402,591 discloses biosynthesis of mevinolin bymicroorganisms belonging to the Pleurotus genus, e.g., Pleurotusostreatus, P. sapidus, P. saca, at 25-35° C. during 7-14 dayscultivation time on surface or submerge cultures.

Canadian Patent No. 2,129,416 discloses the preparation of mevinolin, orin a particular case, mevastatin, with a microorganism belonging to theConiothyrium genus, e.g., under the deposited number Coniothyriumfuckelii ATCC 74227 on a culture medium containing 3-15% glucose, 0.54%peptone, 0.5-5% amylase, 0.2-1% ammonium sulphate, 0.01-0.1% magnesiumsulphate, 0.05-0.2% antifoaming agent, 0.2-1.5% L-isoleucine, 0.2-1.5%L-aspartic acid in the pH range of 5-6. According to the examples theactive ingredient concentration of the broth was within 19-430 mg/liter.

Hungarian Patent No. HU 208,997 discloses the application of theholotype strain Aspergillus obscurus numbered as MV-1, deposited underthe number NCAIM(P)F 001189. The fermentation is preferably carried outon a medium containing yeast extract and/or peptone and/or casein asnitrogen source(s) and glucose and/or maltose or sucrose as carbonsource(s). The activity of the broth at the end of the laboratory scalecultivation is between 400-850 mg/liter.

The foregoing discussion establishes that the development work in thebiosynthesis of mevinolin focused on discovery of newmevinolin-producing microorganisms rather than on the development of thefermentation procedure itself. Several references disclose thatfermentations can be carried out on conventional and known media withthe application of both surface and solid state cultivations. Batch-likeprocedures were applied, where the behaviors of the procedures dependedon the initial conditions. However, technical limitations, e.g.,maintaining the most convenient level of ingredients, optimal dissolvedoxygen supply and pH, etc., made it difficult to implement continuouscorrective actions to ensure more favourable conditions. A givenmicroorganism during the main fermentation stage, depending on itsmetabolism, requires different conditions/composition of media in orderto obtain an optimal growth and production of the active ingredient. Thepresent inventors concluded from their experiments that in the seedculture and at the beginning of the main fermentation, the quantity ofthe active biomass is very small and variable. Thus, the yield of thefermentations are relatively low and variable. Yields reached at the endof the fermentations, which depended of course on the strain, did notexceed a mevinolin concentration of 850 mg/liter. The present inventorsperformed a detailed analysis of the whole fermentation procedure fromthe seed culture stage throughout the end of the fermentation. It wasfound that in the seed culture preparation stage, both in the case ofthe known media and execution processes, the quantity of the biomass istoo low. Therefore, during the main fermentation, the metabolism of themicroorganism and the culture are not adequate.

OBJECTS OF THE INVENTION

It is therefore, one object of the present invention is to improve theefficiency of the mevinolin-producing fermentation procedure by forcingthe production ability of the microorganism via changing the conditionsand the carrying out of the fermentations.

It is another object of the present invention to provide, in ether orboth the seed and main fermentation stage, the most convenient chemicaland physiological conditions for the metabolism by the microorganism.

It is a further object of the present invention to provide, in ether orboth the seed and main fermentation stage, the most convenient chemicaland physiological conditions for the metabolism by the microorganism bymaintaining in a steady state condition, the growth rate and then, foran extended time, a maximal product formation rate.

SUMMARY OF THE INVENTION

These and other objects of the invention are achieved in one embodimentof the present invention by providing a method for producing mevinolinby microorganism in a fermentation process having a seed culture stageand a main fermentation stage, said method comprising:

a) cultivating a microorganism biomass in said seed culture stage toproduce an inoculum;

b) transferring said inoculum into a fermentation medium in said mainfermentation stage; and,

c) maintaining steady stage conditions in said main fermentation stage,thereby producing a fermentation broth containing mevinolin.

In a preferred embodiment of the present invention, steady stateconditions are maintained in the main fermentation stage by one or moreof feeding of organic carbon sources; controlling glucose and/or totalreducing sugar content; feeding of organic and/or inorganic nitrogensources; controlling pH; controlling foam level; controlling the mass ofthe fermentation broth by withdrawals and feedings; and, controlling thedissolved oxygen level. Preferably, the fermentation process inconducted in a submerged culture of the microorganism and at atemperature in the range of from about 24° C. to 30° C. In aparticularly preferred embodiment of the present invention, themicroorganism is an Aspergillus species. In yet other preferredembodiments of the present invention, the organic carbon source isselected from the group consisting of glucose, hydrolyzed starch andvegetable oil; the glucose content is maintained at bellow about 0.2%from the 60th hour of the main fermentation stage; the nitrogen sourcesare selected from the group consisting of corn steep liquor and ammoniumhydroxide; pH is controlled to be within the range of from about 5.2 toabout 7.0, preferably from about 5.2 to about 6.2, feeding carbonsources and/or base; foam level is controlled by addition of a materialfor controlling the foam level, the material preferably being asynthetic material or vegetable oil; and, dissolved oxygen level iscontrolled preferably by stirring and/or aeration of the fermentationbroth. In yet another preferred embodiment of the present invention, theinoculum is transferred from the seed culture stage to the mainfermentation stage when the pH of the seed culture stage is increasingafter having reached its minimum value.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered that optimal biosynthesis of mevinolin maybe performed by adjusting one or more of certain process parameters,steps and/or variables in ether or both the seed culture and mainfermentation stages of the biosynthesis process.

During the seed culture phase, the inventors have found these processparameters, steps and/or variables to include supplying themicroorganisms with the necessary medium components in easilyassimilable form and in the most convenient concentration and, extendingthe cultivation time by about 10 to about 25%.

In order to obtain a steady state condition during the main fermentationstage, the inventors have found these process parameters, steps and/orvariables to include controlling the glucose and/or the total reducingsugar content, maintaining the carbon sources at a suitable minimumlevel, feeding organic and/or inorganic nitrogen sources, controllingpH, controlling foam level, controlling the mass of the broth bywithdrawals and feeding and controlling the dissolved oxygen level bychanging the stirring rate and/or aeration rate.

In order to optimize mevinolin biosynthesis, it is not necessary thateach of the above-mentioned process parameters, steps and/or variablesfor either the seed culture phase or for the main fermentation stage besimultaneously adjusted. However, in a preferred embodiment of thepresent invention, the biosynthesis of mevinolin will involve each ofthe above-mentioned process parameters, steps and/or variables. In sucha preferred embodiment, an advanced metabolic controlled mevinolinfermentation procedure can be carried out in which a steady statecondition, i.e. constant pH, glucose concentration, dissolved oxygen,viscosity, volume, etc., can be reached quickly and which can bemaintained for a long time providing a yield highly exceeding theresults of the known procedures.

Certain advantages may be realized in the seed culture stage byadjustment of one or more of the above-mentioned process parameters,steps and/or variables in that stage. These advantages include, e.g.,reduction of the time requirement to reach the “steady state” conditionby about 20-30% by increasing the number of growth centres and, growthof the active biomass in a more advantageous morphology form, resultingin more favourable conditions for cultivation of the microorganism. As aresult of these advantages and the elongated cultivation time, theconcentration of the active biomass is almost doubled.

Certain advantages may be realized in the main fermentation stage byadjustment of one or more of the above-mentioned process parameters,steps and/or variables in that stage. These advantages include, e.g., afaster and less fluctuating growth phase and quick formation of a steadystate stage that can be maintained for a long time. These advantagesresult in a considerably increased activity of the fermentation.

Thus, in one embodiment, the present invention is directed to afermentation procedure for the manufacture of mevinolin with a strainbelonging to the Aspergillus genus in submerged culture at a pH between5.2 and 7.0, at a temperature within 24 and 30° C., on a mediumcontaining assimilable carbon and nitrogen sources, and mineral salts,wherein a metabolic controlled procedure is applied in the mainfermentation phase in order to maintain the culture in a “steady state”stage. In this embodiment, the total reducing sugar is preferablycontrolled. In the source of the main fermentation, organic carbonsources, e.g., glucose, hydrolyzed starch and vegetable oil arepreferably fed. Preferably, the glucose concentration level ismaintained below 0.2% from the 60th hour of the fermentation. In thecourse of the procedure, nitrogen sources such as corn steep liquor andammonium hydroxide solution are fed. The pH is preferably maintained inthe range of from about 5.2-6.2 by feeding carbon source and/or base,e.g., ammonium hydroxide and/or sodium hydroxide. The foam level thefermenter may also be controlled by feeding vegetable oil, e.g.,sunflower oil and/or soya bean oil, and/or synthetic antifoaming agentinto the broth. Dissolved oxygen is preferably controlled changing thestirring rate and/or aeration rate. In the course of the fermentation,one or more withdrawals are carried out.

In a preferred embodiment, the main culture medium is inoculated by aseed culture having the following composition:

Component Amount (w/v %) glucose 2-6 phosphoric acid 0.002-0.006 acidiccasein 0.2-0.8 corn seep liquor 1.5-5   sunflower oil 0.05-0.18polypropylene glycol 0.05-0.18 pancreatin* 0.002-0.008 *4 times activityaccording to Ph.Hg.VII.

Culture medium with the above composition is completed with theregularly applied micro- and macroelemental salts, e.g., inorganic saltsof sodium, potassium, magnesium and iron. The main culture medium isinoculated with a seed culture of which cultivation time is elongated by10-25%. At the transferring stage of the seed culture the pH is in theincreasing phase after its minimum value.

For the fermentation preferably the Aspergillus obscurus strain, itsvariant, or its mutant, or more preferably the Aspergillus obscurus n.sp. Mv-1 holotype strain deposited under the code number NCAIM(P)F001189 is used.

The seed culture is inoculated into the sterile main fermentation mediumwith elongated cultivation time at the increasing phase of the pH afterits minimum value. In the main fermentation stage, a “steady state”condition with the maximal active ingredient production rate can bemaintained for a long time by feeding of the carbon and nitrogen sourcesin order to supply the nutrient demand; controlling the glucoseconcentration to avoid the undesirable thickening of the culture and theexaggerated increase in biomass; controlling the stirring rate andaeration rate according to the oxygen demand; combining foam levelcontrol with the carbon source demand an appropriate material for bothpurposes, e.g., a mixture of a vegetable oil and a synthetic agent;maintaining the pH between the range of from about 5.2-6.2 with thefeeding of carbon source, e.g., glucose syrup, or base; and, carryingout one or more withdrawals, when the maximal working volume of thefermenter is achieved, or when a mevinolin concentration economicalenough to carry out the downstream processing is reached.

By application of these elements, a versatile controllable fermentationprocedure can be obtained, which depending on the life cycle is able toprovide well-conditioned constant surroundings for the microorganism.

In accordance with the preferred embodiment described above, the presentinvention procedures yields exceeding those of known procedures, usesconsiderably less raw material and energy, reduces the quantity ofenvironmentally polluting waste material relative to the unit mass ofactive material, and better utilizes the fermenter.

In the following Examples, biosynthesis of mevinolin in accordance withthe process disclosed in HU 208997 (Comparative Example 1) is comparedwith the process in accordance with a preferred embodiment of thepresent invention (Example 2).

Comparative Example 1

Biosynthesis of Mevinolin in Accordance with HU 208997

A seed culture medium having the following composition is prepared in a600 liter vessel:

Component Amount (%) glucose 4.0 casein peptone 0.5 NaNO₃ 0.3 KH₂PO₄ 0.2KCl 0.05 MgSO₄*7H₂O 0.05 FeSO₄*7H₂O 0.001

Seeding of the inoculum was carried out by the spore suspension of theAspergillus obscurus strain with spore number of 6.5×10⁹. Processparameters of the seed culture were as follows:

Parameter Value Volume 400 liters Temperature 27° C. Aeration Rate 20Normal m³/h Internal Pressure: 0.5 bar Stirring Rate: 320 rpm

Transferring of the seed culture into the main fermentation medium isdone according to the standard procedure at the age of 36 hour, when thepH was in its decreasing stage at 5.6. Centrifuged packed cell volume ofthe biomass (PCV) was 14%.

The above seed culture was inoculated into the main fermentation mediumlabeled MEF-03 at 10% inoculation rate. The composition of the mainfermentation medium was as follows:

Component Amount (%) Dextrose Monohydrate 1.0 Acidic Casein 0.2 CornStarch 8 Soya Bean Meal 1.5 Corn Steep Liquor (50%) 1 Sodium Chloride 1Potassium Dihydrogen Phosphate 0.2 Sodium Glutamate 1.2 Sunflower Oil0.16 Polypropyleneglycol 0.16 Pancreatine* 0.002 BAN 240 Enzyme 0.007CaCl₂ 0.02 Potassium Hydroxide for pH setting *4 times strengthaccording to Ph.Hg.VII.

Volume at 0 hour: 500 liters.

The fermentation is carried out for 7 days. The aeration and stirringrates are as follows:

Aeration* Stirring rate* 18-25 Normal m³/h 180-320 rpm *Dissolved oxygenis maintained above 40% of the saturation value.

During the course of the fermentation, four 50 kg portions enzymaticallyliquefied corn starch is fed according to a schedule at the age of 50,73, 90 and 108 hours.

Data for the Active Ingredient Production

Fermentation time: 164 hours. Activity measured by HPLC: 927 mg/kg.Quantity of the broth: 580 kg. As a consequence of the above, thequantity of the fermented active ingredient is 0.58 ton*927 g/ton/1000/1m³=0.54 kg/m³ total volume.

EXAMPLE 2

Biosynthesis of Mevinolin with Glucose Syrup and Nitrogen Source Feedingand pH Control by Sodium Hydroxide or Ammonium Hydroxide

In a 600 liter vessel is prepared a seed culture medium having thefollowing composition:

Component % glucose 4.0 casein peptone 0.5 corn steep liquor (50%) 3.0NaNO₃ 0.3 KH₂PO₄ 0.2 KCl 0.05 MgSO₄*7H₂O 0.05 FeSO₄*7H₂O 0.001pancreatine* 0.005 polypropyleneglycol 0.1 sunflower oil 0.1 *4 timesstrength according to Ph.Hg.VII.

Seeding was done by the spore suspension of the Aspergillus obscurusstrain with spore number of 6.5×10⁹. Process parameters of the seedculture were the same as set forth in Comparative Example 1.

However, transferring of the seed culture was done differently from thetransferring described in Comparative Example 1. The age of transferringwas 40 hours. The pH reached a minimum value (pH 4.9), and thetransferring was in the stage when the pH had started to increase andreached about 5.0. Thus, the pH had increased about 0.1 from its minimumvalue. The centrifuged packed cell volume of the biomass (PCV) was 24%.

The above seed culture was inoculated into the main fermentation mediumat a 10% inoculation rate. The composition of the main fermentationmedium was the following:

Component Amount(%) corn starch 8 glucose syrup* 1.0 acidic casein 0.2soya bean meal 1.5 corn steep liquor (50%) 1 sodium chloride 1 potassiumdihydrogen phosphate 0.2 sodium glutamate 1.2 sunflower oil 0.16polypropyleneglycol 0.16 pancreatine** 0.002 potassium hydroxide for pHsetting *25 kg was fed in the form of 25% glucose syrup **4 timesstrength according to Ph.Hg.VII.

Fermentation was carried out for 13 days. The aeration and stirringrates are as follows:

Aeration* Stirring rate* min. 12, max. 32 Normal m³/h min. 220, max. 400RPM *Dissolved oxygen was maintained above 40% of the saturation valueby aeration priority method.

In the course of the fermentation, the maintenance of the dissolvedoxygen is very important. In the most intensive stage, this can bereached by the application of about 25-32 Normal m³/h aeration rate and300-400 RPM stirring rate.

During the main fermentation stage, the temperature was 27±2° C., theinner pressure was 0.4 bar, and the cultivation time was 309 hours.

In the course of the fermentation, the following nutrients were fed:

1. Hydrolyzed corn starch (glucose syrup) is prepared with enzymatic andhydrochloric acid treatment and fed. The raw materials used to preparethe glucose syrup were as follows: 25% corn starch, 0.3-0.4% CaCl₂,0.1-0.2% amylase enzyme (BAN) and, 1% concentrated hydrochloric acid.Feeding of the glucose syrup started in the increasing stage of the pH,after its minimum value (5.0) in the age of 45 hours and at the pH valueof 5.6. Feeding was carried out in a continuously, maintaining the pH inthe range of 5.4 and 5.8. The minimum rate of feeding was 0.5 liter/hourand the maximal rate of feeding was 5 liter/hour.

2. NaOH or NH₄OH solution was feed for pH control when the pH droppedbelow 5.5 in addition to the minimum feeding rate of the glucose syrup.

3. Corn seep liquor (1%) was fed at the age of 100 hours of thefermentation (related to the 0 hour volume).

Data for the Active Ingredient Production

Fermentation time: 309 hours. Activity measured by HPLC: 2868 mg/kg.Quantity of the broth: 680 kg. As a consequence of the above, thequantity of the fermented active ingredient is 0.68 ton*2868g/ton/1000/1 m³=1.95 kg/m³ total volume.

EXAMPLE 3

Biosynthesis of Mevinolin with Feedings, Controlling and Withdrawals

Aspergillus obscurus n. sp. MV-1 holotype strain is cultivated on asterile inoculum medium with the following composition:

Component % glucose 4.0 phosphoric acid 0.0035 acidic casein 0.5 cornsteep liquor (50%) 3.0 NaNO₃ 0.3 KH₂PO₄ 0.2 KCl 0.05 MgSO₄*7H₂O 0.05FeSO₄*7H₂O 0.001 sunflower oil 0.1 polypropylene glycol 0.1 pancreatine*0.005 potassium hydroxide for pH setting hydrochloric acid for pHsetting *4 times strength according to Ph.Hg.VII.

The following process parameters were employed during the inoculumstage:

Parameter Value Volume 8 m³ Stirring rate 120 RPM Aeration 400 ± 50Normal m³/h Inner pressure 0.4 ± 0.1 bar Temperature 27 ± 2 ° C.

The inoculum was transferred after its pH had reached its minimum value(pH 4.8) and when the value was 0.1 unit above the minimum, i.e. whenthe pH had reached a value of 4.9. The centrifuged packed cell volume ofthe biomass (PCV) was 24%. At an 8% transfer rate, the above inoculumwas transferred into a main fermentation medium having the followingcomposition:

Component Amount (%) Soya bean meal 1.28-1.57 sum of corn or wheatstarch 9 acidic casein 0.20 corn steep liquor (50%) 0.857-1.14  sodiumchloride 1.0 potassium dihydrogen phosphate 0.2 sodium glutamate1.14-1.20 calcium chloride 3.8 × 10⁻³ BAN enzyme 2.2 × 10⁻³ sunfloweroil 0.10 polypropyleneglycol 0.10 pancreatine* 2.0 × 10⁻³ potassiumhydroxide or hydrochloric acid for pH setting *4 times strengthaccording to Ph.Hg.VII.

The following process parameters were employed during the mainfermentation stage:

Parameter Value Inner pressure 0,2 ± 0,05 bar Temperature 27 ± 2 ° C.Stirring rate 60-85 RPM Aeration rate 1000-4000 Normal m³/h

Feeding Materials

1. Carbon source material: About 40 ton hydrolyzed starch, degraded to alarge extent until glucose (glucose syrup) is prepared in 25% form isfed continuously. The raw materials used to prepare the glucose syrupare as follows: 25% corn or wheat starch, 0.3 calcium chloride, 0.3% BAN240 enzyme and about 2% hydrochloric acid.

2. Nitrogen source material: Corn steep liquor (50%) is used for thepreparation in 1% quantity related to 0 hour fermentation volume in 5 m³sterile volume.

3. Base for pH control. Unsterilized 25-30% sodium hydroxide or ammoniasolution is used for pH control.

Feeding

1. Glucose syrup: Feeding is started at about 50 hours in the increasingstage of the pH after its first minimum value. Glucose syrup is fed inorder to control the pH in the range of 5.4 and 5.8. Glucose feeding iscarried out in dose form or continuously. The glucose syrup is fed at arate in the range of 0-1000 kg/hour, preferably in the range of 150-500kg/hour. When the pH minimum could not be maintained, even with theminimum glucose syrup feeding rate, base feeding was necessary tocontrol pH.

2. Corn steep liquor: The corn steep liquor is fed in one dose at about100 hour. Further doses could be fed if necessary.

3. NaOH or NH₄OH: The base was fed and pH control was performed when, inaddition to the minimum glucose syrup feeding rate, the pH droppedbellow 5.5.

The foam level is controlled by the feeding of the mixture of sunfloweroil:PPG in the rate of 95:5 in order not to exceed a foam volume of 25%of the fermenter working volume.

Withdrawals were carried out from the fermenter at the ages of 112, 138,178, 204 hours by harvesting 4 m³ broth 4 times.

Data for the Active Ingredient Production

Fermentation time: 226 hours. Activity measured by HPLC (mother part):1825 mg/kg. Harvested activity (with withdrawals): 1788 mg/kg. As aconsequence of the above, the quantity of the fermented activeingredient is 95 ton*1788 g/ton/1000/105 m³ total volume.

What is claimed is:
 1. A fermentation procedure for the manufacture oflovastatin with a strain belonging to the Aspergillus genus in submergedculture at a pH between 5.2 and 7.0, at a temperature within 24 and 30°C., on a medium containing assimilable carbon and nitogen sources, andmineral salts, comprising a seed culture stage and a main fermentationstage wherein a metabolic controlled procedure is applied in the mainfermentation stage in order to maintain the culture in a steady state.2. A process of claim 1, wherein the metabolic controlled procedurecomprises controlling total reducing sugar content during the mainfermentation stage.
 3. A process of claim 1, wherein organic nitrogensource is fed in the course of the main fermentation phase.
 4. A processof claim 3, wherein as a carbon source glucose is fed.
 5. A process ofclaim 4, wherein the glucose content is controlled by feeding from theage of 60 hours till the end of the fermentation below 0.2%.
 6. Aprocess of claim 3, wherein as a carbon source hydrolysed starch and/orvegetable oil are fed.
 7. A process of claim 1, wherein nitrogen sourceis fed in the course of the main fermentation phase.
 8. A process ofclaim 7, wherein as a nitrogen source corn steep liquor and/or ammoniumhydroxide are fed.
 9. A process of claim 1, wherein the pH is controlledin the course of the main fermentation phase between 5.2 and 6.2.
 10. Aprocess of claim 9, wherein the pH control is done by the carbon sourceand/or base feeding.
 11. A process of claim 10, wherein ammoniumhydroxide is used for base feeding.
 12. A process of claim 1, furthercomprising a step of controlling foam formation.
 13. A process of claim12, wherein the foam control is done by vegetable oil and syntheticagent.
 14. A process of claim 13, wherein the vegetable oil is sunfloweroil and/or Soya bean oil.
 15. A process of claim 1, wherein the stirringrate and/or aeration rate are controlled according to the dissolvedoxygen level.
 16. A process of claim 1, wherein partial harvests aredone in the course of the main fermentation phase.
 17. A process ofclaim 1, wherein the inoculation of the main fermentation phase is doneby the Aspergillus microorganism culture cultivated on the followingseed culture medium: glucose 2-6 w/v % phosphoric acid 0.002-0.006 w/v %acid casein 0.2-0.8 w/v % corn seep liquor, 50% 1.5-5 w/v % sunfloweroil 0.05-0.18 w/v polypropylene glycol 0.002-0.008 w/v % pancreatin, 4times activity according to Ph.Hg. VII

completed with water and the regularly applied micro- and macroelementalsalts.
 18. A process of claim 1, wherein the transfer of the seedculture into the main fermentation is carried out by the application ofthe 10-25% elongated cultivation time related to the regular one and/orin the increasing phase of the pH after its minimum value.
 19. A processof claim 1, wherein the applied microorganism is Aspergillus obscurus.20. A process of claim 19, wherein the applied microorganism for thefermentation is the Aspergillus obscurus n. sp. MV-1 holotype straindeposited under the code number NCAIM(P)F 001189.